Method of stabilizing protein C or activated protein C and the stabilized composition obtained by said method

ABSTRACT

PCT No. PCT/JP94/01804 Sec. 371 Date Apr. 23, 1996 Sec. 102(e) Date Apr. 23, 1996 PCT Filed Oct. 27, 1994 PCT Pub. No. WO95/11698 PCT Pub. Date May 4, 1995A method for stabilizing protein C or activated protein C and a preparation obtained by said method are provided, said method and preparation being applicable during procedures such as isolation and purification, lyophilization, heating, etc. or when stored. To a salt buffer containing protein C or activated protein C and sodium ion are added at least one amino acids, and further either one or a combination of albumin and a non-ionic surfactant.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for stabilizing protein C oractivated protein C which is derived from plasma or is prepared by usingthe genetic recombination technique. More particularly, the presentinvention relates to a method for stabilizing protein C or activatedprotein C when it is stored or subjected to procedures such as isolationand purification, lyophilization, treatment by heating, etc. and to apreparation stabilized by said method.

TECHNICAL BACKGROUND

Protein C (hereinafter also referred to as "PC") is a kind of a vitaminK dependent protein, i.e. a protein containing γ-carboxyglutamic acid,and is activated to activated protein C (hereinafter also referred to as"APC") by thrombin in the presence of thrombomodulin present on thesurface of the vascular endothelial cell. Activated protein C is a kindof a serine protease and exhibits a strong anti-coagulant activity byinactivating cofactors of the blood coagulation system such as Factor Va(FVa) and Factor VIIIa (FVIIIa). It is also known that activated proteinC releases a plasminogen activator from the vascular wall to acceleratethe fibrinolytic system. Furthermore, it is known that a defect inprotein C causes a severe thrombosis. Thus, it has been established thatactivated protein C is the most important factor which regulates theblood coagulation and fibrinolytic system. Therefore, protein C oractivated protein C is expected to be exploited as a novelanti-coagulating agent or profibrinolytic agent.

It has hitherto been known that the amount of protein C present in aplasma or expressed in a tissue culture system is extremely low.Accordingly, in order to use protein C or activated protein C as ananti-coagulant agent or a profibrinolytic agent widely and safely,isolation and purification of protein C or activated protein C isimportant. In addition, storage as a solution or in frozen form for longperiods of time, lyophilization or procedures for inactivation ofcontaminating viruses such as heating are indispensable to the processwhen protein C or activated protein C is industrially prepared on alarge scale. However, storage, freezing or freeze-drying, or heattreatment of a highly pure protein C or activated protein C extremelylowers the activity thereof. There has also been no report on thestability of highly purified protein C or activated protein C. Undersuch circumstances, it is impossible to provide a highly pure protein Cor activated protein C efficiently and stably on an industrial scale.

Under such circumstances, the present inventors have earnestly studiedthe stability of protein C or activated protein C, and as a result, havefound that the activity of protein C or activated protein C can bemaintained even after storage for a significant period of time or afterprocedures such as isolation and purification, lyophilization, heating,etc. by adding, to protein C or activated protein C, a salt buffer suchas phosphate or citrate buffer containing sodium ion supplemented withat least one amino acid, and further adding either one or a combinationof albumin and a non-ionic surfactant, and thereby, the presentinvention has been completed.

DISCLOSURE OF THE INVENTION

The present invention relates to a method for stabilizing protein C oractivated protein C which comprises adding, to a salt buffer containingprotein C or activated protein C and sodium ion, at least one aminoacid, and further either one or a combination of albumin and a non-ionicsurfactant. More particularly, the present invention relates to a methodfor stabilizing protein C or activated protein C which comprisesdissolving protein C or activated protein C in a salt buffer such asphosphate or citrate buffer containing sodium ion, and adding to saidbuffer at least one of amino acids which constitute a protein, e.g.glycine, alanine, lysine, arginine, aspartic acid, glutamic acid, etc.and a polypeptide having a protein-stabilizing activity such as albumin,globulin, etc. and, in a preferable embodiment, optional non-ionicsurfactant, typically Tween 80 is added. The invention also relates topreparations stabilized by said method.

BEST MODE FOR CARRYING OUT THE INVENTION

Protein C or activated protein C used herein encompasses variants orderivatives thereof which substantially has APC activity and may beprepared by the known methods, for example, by preparing protein C byisolation from human plasma or production utilizing the geneticrecombination technique and then activating protein C; by directlyisolating APC from human blood; or by producing APC by the use of thegenetic recombination technique, etc. Activation of protein C into APCcan be carried out by any known method, for example, by activation withthrombin isolated from human or bovine blood, or by activation with anequivalent protease, etc.

Production of APC derived from blood can be conducted, for example, byactivating protein C, which is purified from human plasma by affinitychromatography using an anti-protein C antibody, with human thrombin,and purifying the resulting activated protein C by a cationchromatography (Blood, 63, p.115-121 (1984)); or by activating protein Cwhich is purified from human plasma by barium citrate adsorption andelution, fractionation with ammonium sulfate, DEAE-Sephadex columnchromatography, dextran sulfate agarose chromatography andpolyacrylamide gel electrophoresis, etc. to produce APC in accordancewith the method described by Kisiel (J. Clin. Invest., 64, p.761-769(1979)); or by activating a commercially available blood-coagulatingpreparation containing protein C to produce APC in accordance with themethod described by Taylor et al. (J. Clin. Invest., 79, p.918-925(1987)), and the like.

Production of APC utilizing the genetic recombination technique can beconducted, for example, in accordance with the methods described inJapanese Patent First Publication (Kokai) No. 61-205487, Japanese PatentFirst Publication (Kokai) No. 1-2338 or Japanese Patent FirstPublication (Kokai) No. 1-85084, etc. A process for preparing thestarting material protein C or activated protein C as used herein is notlimited to the above-mentioned procedures.

The thus prepared starting material protein C or activated protein C isisolated and purified by a combination of usual biochemical proceduresfor isolation and purification, including, for example, a salting-outwith ammonium sulfate, an ion-exchange chromatography with an ionexchange resin, gel filtration, electrophoresis, etc.

When the degree of purification is increased by such procedures, proteinC or activated protein C is liable to become unstable. Even a producthaving not so much high purity also shows a decrease in the activity dueto the procedures such as storage, freezing, lyophilization, heating,etc. The present invention is primarily aimed at the stabilization ofsuch protein C or activated protein C which became unstable with theincrease of purification degree. Protein C or activated protein C to bestabilized in accordance with the present invention may be either in theform of solution or powder.

In the stabilization method for protein C or activated protein C of thepresent invention, a salt containing as a stabilizing agent sodium ionat a concentration of preferably 50 mM to 200 mM, at least one aminoacid and a polypeptide having a stabilizing effect are added to a buffercontaining 100 to 2500 U/ml of protein C or activated protein C. Thesalt and amino acid may be used each alone or in a combination of two ormore thereof. Preferable buffer includes, for example, sodium citrate,sodium phosphate, and sodium sulfate, etc. The amino acid is added at afinal concentration of 0.005 M to 0.1 M, more preferably 0.01 M to 0.05M. A polypeptide having a stabilizing effect such as albumin or globulinis added at an appropriate concentration which may be determined basedon a common sense or from the economical point of view, preferably at aconcentration of 0.5% (W/V) to 10% (W/V). The unit "% (W/V)" as usedherein denotes an amount of a solute dissolved in one liter of asolution, for example, when 10 g of a solute is dissolved in one literof a solution, a concentration is 1% (W/V). In a preferable embodimentof the present invention, a non-ionic surfactant such as Tween 80 mayoptionally be added at a concentration of 0.0005% (W/V) to 0.1% (W/V) toaccelerate the stabilization effect.

A typical embodiment of the present invention is an aqueous buffersolution containing protein C or activated protein C, which comprises100 to 2500 U/ml of protein C and/or activated protein C, 50 to 200 mMof sodium ion, 5 to 100 mM of an amino acid, and further either one or acombination of 0.5 to 10% (W/V) of albumin and 0.0005 to 0.1% (W/V) of anon-ionic surfactant.

When a stabilizing agent is added to protein C or activated protein C inthe form of powder, it is used in such an amount that the concentrationof the stabilizing agent becomes in the above-mentioned range when saidpowder is dissolved.

Thus, in another typical embodiment of the present invention, acomposition containing protein C or activated protein C comprises 1×10⁵to 2.5×10⁶ U of protein C and/or activated protein C, 50 to 200 mg ofsodium ion, 5 to 100 millimoles of an amino acid, and further either oneor a combination of 5 to 100 g of albumin and 0.005 to 1 g of anon-ionic surfactant.

The manner of addition of these ingredients may not be specified butincludes various methodes, for example, by adding directly powderymaterials of the present invention to a buffer solution containingprotein C or activated protein C; or by previously dissolving saidpowdery materials in water or a suitable buffer and adding the solutionto the buffer solution containing protein C or activated protein C; orby mixing said powdery materials with a protein C- or activated proteinC-containing powder. Addition may be carried out either during theprocess of isolation and purification of said protein or the process forproducing a pharmaceutical preparation.

When a solution containing protein C or activated protein C added withthe stabilizing agent of the present invention is stored, or subjectedto procedures such as isolation and purification, or process forproducing a pharmaceutical preparation in the state of solution, it ispreferably done at 0 to 30° C., more preferably at 0 to 10° C. When saidsolution is stored in a freezed state, it is preferably done at a lowertemperature than the freezing point, more preferably at lower than -20°C., or when it is stored in a lyophilized state, it is preferably doneat room temperature or lower. By using the solution containing protein Cor activated protein C incorporated with the stabilizing agent of thepresent invention, the activity of protein C or activated protein C canbe stably maintained even during storage in the state of a solution, orin a freezed or lyophilized state, or even in the treatment thereof suchas isolation and purification or process for producing a pharmaceuticalpreparation.

The activity of APC was measured in accordance with the followingprocedures.

One unit of APC activity is defined as an amount of APC which prolongstwice an activated thromboplastin time (APTT; second) of normal humanplasma. Accordingly, the activity of APC is measured wherein APTT inseconds is measured for normal human plasma to which a diluted sample isadded and the dilution at which the measured APTT value is twice that ofcontrol (buffer) is determined and regarded as the activity of APC forsamples.

(Procedures)

A sample is diluted with a veronal buffer containing 1% human serumalbumin to, for example, 400 times, 500 times, 800 times or 1000 timesdilution. To each 100 μl of either control (buffer) or samples of eachdilution are added 100 μl of normal human plasma (e.g. Citrol I: BaxterDiagnostics Inc.) 100 μl and APTT reagent (e.g. Actin: BaxterDiagnostics Inc.) 100 μl at 37° C. successively with an interval of 15seconds, the mixture is stirred, and after 2 minutes, 0.025 M CaCl₂ 100μl is added and the coagulation time is measured.

(Calculation of Activity)

A linear regression formula and a correlation coefficient of 10 ³ /X andY are obtained from the APTT values (Y) at each dilution (X) of thecontrol and the samples as follows:

    Y=A(10.sup.3 /X)+B

A value of X₁ obtained from the following formula:

    X.sub.1 =10.sup.3 {(Y.sub.1 -B)/A}

wherein Y₁ is a value twice that of the APTT (second) of control, isregarded as the activity of APC (U/ml) for samples.

The activity of protein C was measured by using "Staclot Protein C"manufactured by Boehlinger Mannheim.

The present invention is illustrated in more detail by means of thefollowing Examples but should not be construed to be limited thereto.

EXAMPLE 1

Effect of Various Counterions on Stability of APC:

To a solution containing human activated protein C with the activity of500 U/ml was added 2.5% human serum albumin (hereinafter alsoabbreviated as "HSA"). The solution was then dialyzed against a solutionof Na citrate, Na phosphate and Na sulfate (each 20 mM), containing 0.7%NaCl and 0.067 M glycine. After dialysis, each solution was left tostand at 37° C. for 24 hours and the activity was measured. The resultsare shown in Table 1. All the counterions tested, Na citrate, Naphosphate and Na sulfate, showed a similar satisfactory stability.

                  TABLE 1    ______________________________________    Effect of various counterions on stability of APC                   Rate of remaining activity (%)    Counterion     (37° C., 24 hours)    ______________________________________    Na citrate 20 mM                   97.4    Na phosphate 20 mM                   94.8    Na sulfate 20 mM                   92.2    ______________________________________

EXAMPLE 2

Effect of Amino Acids on Stability of APC:

To a solution containing activated protein C with the activity of 500U/ml was added 2.5% HSA. The solution was then dialyzed against asolution of sodium citrate buffer containing 0.7% NaCl and each 0.05 Mof either glycine, alanine, lysine, arginine, aspartic acid or glutamicacid. After dialysis, each solution was left to stand at 37° C. for 24hours and the activity was measured. The results are shown in Table 2.All the above six amino acids showed a high stability withoutdeteriorating the stability of APC.

                  TABLE 2    ______________________________________    Effect of various amino acids on stability of APC    Amino Acid     Rate of remaining activity (%)    (0.5%)         (37° C., 24 hours)    ______________________________________    No amino acid  86.1    Gly            97.7    Ala            97.7    Lys            107    Arg            102    Asp            108    Glu            106    ______________________________________

EXAMPLE 3

Effect of Addition of HSA:

Solutions containing human activated protein C (1700 U/ml), 20 mMcitrate, 0.7% NaCl and 0.067 M glycine with and without addition of 2.5%HSA were prepared. The solutions were then left to stand at 37° C. and4° C. The activity was measured with the passage of time in accordancewith the method described herein and a retention percent of activity wasobtained. The results are shown in Table 3.

                  TABLE 3    ______________________________________    Retention percent of activity under respective    conditions (%)    ______________________________________    At 37° C.:           Retention time (hour)             0      1        3    6      8    24    ______________________________________    HSA (+)  100    --       99.2 --     101  97.4    HSA (-)  100    88.8     92.0 86.2   90.9 79.3    ______________________________________    At 4° C.:           Retention time (day)             0      1        3    5      7    14    ______________________________________    HSA (+)  100    --       105  --     --   98.1    HSA (-)  100    112      99.1 104    98.9 85.2    ______________________________________

As will be understood from the results shown in Table 3, the systemwithout HSA can not prevent decrease in the activity of APC when it isleft standing. From these results, it is concluded that 2.5% HSAextremely stabilizes APC.

EXAMPLE 4

Effect of HSA on Stability of APC:

The data shown in Table 4 indicates that the activity of APC isdependent on the concentration of HSA. To a solution of activatedprotein C (500 U/ml) was added 0.5 to 10.0% HSA. This solution was putin a storage vessel and left to stand at 37° C. After 24 hours, a samplewas taken and the activity was measured. In case of no addition of HSA,the activity was decreased by about 20% whereas in case of addition ofHSA at a concentration ranging from 0.5 to 10.0%, almost no decrease inthe activity was observed and APC remained stable.

                  TABLE 4    ______________________________________    Effect of HSA on stability of APC activity               Rate of remaining activity (%)    HSA (%)    (37° C., 24 hours)    ______________________________________    0.0        79.0    0.5        96.0    2.5        97.4    5.0        98.0    10.0       102    ______________________________________

EXAMPLE 5

Effect of Tween 80 on Stability of APC:

To a solution containing human activated protein C (500 U/ml), 20 mMcitrate, 0.7% NaCl and 0.067 M glycine was added 0.0005% to 0.1%non-ionic surfactant, Tween 80 (trade name). This solution was put in astorage vessel and left to stand at 37° C. After 24 hours, a sample wastaken and the activity was measured. The results are shown in Table 5.In case of no addition of Tween 80, the activity was decreased by about20% and the stability was deteriorated whereas in case of addition ofTween 80 at a concentration ranging from 0.0005 to 0.1%, no change inthe activity was found and the solution maintained high stability.

                  TABLE 5    ______________________________________    Tween 80   Rate of remaining activity (%)    (%)        (37° C., 24 hours)    ______________________________________    No          79    addition    0.0005     101    0.025      104    0.1        103    ______________________________________

EXAMPLE 6

Effect of NaCl on Quality of Lyophilized Solid Citrate:

An APC lyophilized preparation containing APC 500 U/ml, HSA 2.5%, Gly0.067 M and Na citrate 20 mM was prepared such that it contains sodiumchloride at a concentration ranging from 1 mM to 500 mM. Eachlyophilized preparation was stored at 60° C. for a month and theappearance quality of the solids was observed as shown in Table 6.

                  TABLE 6    ______________________________________    Quality of lyophilized preparation containing NaCl    at a selected concentration           Quality of solids    Na chloride              After One month storage    (mM)     Pre-storage     at 60° C.    ______________________________________     1       White solid, shrink                             Extremely shrink     10      White solid, a bit shrink                             Same as pre-storage     50      White porous lump                             Same as pre-storage    100      White porous lump                             Same as pre-storage    500      Glassy shrunk lump                             Same as pre-storage    ______________________________________

As is clear from the results shown in Table 6, a solids APC preparationis difficult to formulate into a pharmaceutical preparation in the caseof addition of sodium chloride both at a higher and lower concentration.The presence of sodium chloride at a concentration of 50 mM to 200 mM isconsidered to contribute to the stabilization of the lyophilizedpreparation.

EXAMPLE 7

APC Activity in Lyophilized Preparation:

A citrate buffer solution containing the stabilizing agent of thepresent invention (0.7% NaCl, 0.067 M glycine and 2.5% HSA) was preparedso that it contains the APC activity at 100 to 2500 U/vial and sterilelydivided into vials, which were lyophilized and sealed. Each vial wasleft to stand at 10° C., 15° C. and 60° C. and decrease in the activitywas determined. The results are shown in Table 7. The data show that themethod for stabilization of APC of the present invention is effective inthe state of lyophilization.

                  TABLE 7    ______________________________________    Rate of remaining activity (%)    APC    (U/vial) Time     10° C.                                  15° C.                                        60° C.    ______________________________________     100     30 months                      100         101   --     500     30 months                      97          99    --             11 days                     99    1000     30 months                      99          97    --    2500     30 months                      98          95    --             11 days                    105    ______________________________________

EXAMPLE 8

Effect Repeated Freezing-melting on the Stability of APC:

To a citrate buffer solution containing activated protein C with theactivity of 500 U/ml was added the stabilizing agent of the presentinvention (0.7% NaCl, 0.067 M glycine and 2.5% HSA). The resultingsolution was subjected to repetition (5 times, 10 times, 15 times and 20times) of freezing (-80° C.) and melting and the activity was measured.The results are shown in Table 8. In spite of 20 repetitions offreezing-melting, the activity of APC showed no significant change andremained stable.

                  TABLE 8    ______________________________________    Repetition Rate of maintaining activity (%)    ______________________________________     5         102    10         95.1    15         106    20         99.2    ______________________________________

What is claimed is:
 1. A method for stabilizing protein C or activatedprotein C which comprises adding a salt buffer, a sodium salt for afinal concentration of 50 to 100 mM of sodium ion, at least one aminoacid for a final concentration of 0.005M to 0.1M, and a compoundselected from the group consisting of albumin for a final concentrationof 0.5 to 10% w/v, a non-ionic surfactant for a final concentration of0.0005 to 0.1% w/v, and a mixture thereof, to protein C or activatedprotein C to form a protein C or activated protein C-containingcomposition in which protein C or activated protein C is stable at 37°C. for 24 hours.
 2. The method for stabilizing protein C or activatedprotein C of claim 1, wherein said at least one amino acid is selectedfrom the group consisting of natural occurring α-amino acids.
 3. Themethod for stabilizing protein C or activated protein C of claim 2wherein said at least one amino acid is selected from glycine, alanine,lysine, arginine, aspartic acid, glutamic acid, and mixtures thereof. 4.An aqueous buffer solution containing protein C and/or activated proteinC which contains 100 to 2500 U/ml of protein C and/or activated proteinC, 50 to 200 mM of sodium ion, 5 to 100 mM of an amino acid, and furthereither one or a combination of 0.5 to 10% (w/v) of albumin and 0.0005 to0.1% (w/v) of a non-ionic surfactant, wherein said protein C and/oractivated protein C is stable in said aqueous buffer solution at 37° C.for 24 hours.
 5. A composition containing protein C and/or activatedprotein C which contains 1×10⁵ to 2.5×10⁶ U of protein C and/oractivated protein C, 50 to 200 mg of sodium ion, 5 to 100 millimoles ofan amino acid, and further either one or a combination of 5 to 100 g ofalbumin and 0.005 to 1 g of a non-ionic surfactant, wherein said proteinC and/or activated protein C is stable in said composition at 37° C. for24 hours.
 6. The method for stabilizing protein C or activated protein Caccording to claim 1, wherein said adding step comprises firstdissolving said protein C or activated protein C in said salt buffer,and then subsequently adding said sodium ion, said at least one aminoacid, and said compound thereto.
 7. The aqueous buffer solutionaccording to claim 4, wherein 0.0005% to 0.1% w/v of a non-ionicsurfactant is present in said aqueous buffer solution.
 8. The aqueousbuffer solution according to claim 4, wherein 0.0005 to 10% w/v of anon-ionic surfactant in combination with 0.5 to 10% w/v of albumin arepresent in said aqueous buffer solution.
 9. The composition according toclaim 5, wherein 0.005 to 1 g of a non-ionic surfactant is present insaid composition.
 10. The composition according to claim 5, wherein0.005 to 1 g of a non-ionic surfactant in combination with 5 to 100 g ofalbumin are present in said composition.
 11. A method for stabilizingprotein C or activated protein C which comprises adding a salt buffer,sodium ion, at least one amino acid, and a compound selected from thegroup consisting of a non-ionic surfactant and a mixture of a non-ionicsurfactant and albumin to protein C or activated protein C to stabilizeprotein C or activated protein C.
 12. The method according to claim 11,wherein said at least one amino acid is added to a final concentrationin a range of 0.005M to 0.1M.
 13. The method according to claim 11,wherein said at least one amino acid is a naturally occurring α-aminoacid.
 14. The method according to claim 13, wherein said at least oneamino acid is selected from glycine, alanine, lysine, arginine, asparticacid, glutamic acid, and mixtures thereof.
 15. The method according toclaim 11, wherein said non-ionic surfactant is added to a finalconcentration in a range of 0.0005% w/v to 0.1% w/v.
 16. The methodaccording to claim 11, wherein said albumin is added to a finalconcentration in a range of 0.5% w/v to 10% w/v.
 17. The methodaccording to claim 11, wherein said adding step comprises firstdissolving said protein C or activated protein C in said buffer, andsubsequently adding said sodium ion, said at least one amino acid, andsaid compound thereto.
 18. The method according to claim 1, furthercomprising the step of lyophilizing the protein C or activated proteinC-containing composition.
 19. The method according to claim 18, whereinprotein C or activated protein C in the lyophilized composition isstable at 15° C. for 30 months or at 60° C. for 11 days.